Aloe-emodin induced in vitro G2/M arrest of cell cycle in human promyelocytic leukemia HL-60 cells

doi:10.1016/j.fct.2004.03.002

Food and Chemical Toxicology

Volume 42, Issue 8, August 2004, Pages 1251-1257

https://doi.org/10.1016/j.fct.2004.03.002 Get rights and content

Abstract

In this study, we have evaluated the chemopreventive role of aloe-emodin in human promyelocytic leukemia HL-60 cells in vitro by studying the regulation of proliferation, cell cycle and apoptosis. Aloe-emodin inhibited cell proliferation and induced G2/M arrest and apoptosis in HL-60 cells. Investigation of the levels of cyclins B1, E and A by immunoblot analysis showed that cyclin E level was unaffected, whereas cyclin B1 and A levels increased with aloe-emodin in HL-60 cells. Investigation of the levels of cyclin-dependent kinases, Cdk1 and 2, showed increased levels of Cdk1 but the levels of Cdk2 were not effected with aloe-emodin in HL-60 cells. The levels of p27 were increased after HL-60 cells were cotreated with various concentrations of aloe-emodin. The increase of the levels of p27 may be the major factor for aloe-emodin to cause G2/M arrest in these examined cells. Flow cytometric assays and DNA fragmentation gel electrophoresis also confirmed aloe-emodin induced apoptosis in HL-60 cells. The levels of caspase-3 were increased after HL-60 cells were cotreated with 10 μM aloe-emodin for 12, 24, 48, and 72 hours. Taken together, aloe-emodin therefore appears to exert its anticarcinogenesis properties by inhibiting proliferation and inducing cell cycle arrest and apoptosis underwent activation of caspase-3 in human leukemia HL-60 cells.

Introduction

Leukemia of human blood has poor therapy. Therapeutic approaches for human leukemia include radiation treatment, hyperthermia and chemotherapy. However, conventional strategies for treatment of human leukemia are not yet satisfactory. Some of the well known anticancer drugs are known to induce apoptosis via the inhibition of topoisomerase II (Kaufmann, 1989; Walker et al., 1991; Ling et al., 1993; Stevnsner and Bohr, 1993). Cell apoptosis is characterised and involved by a series of typical morphological events, for example shrinkage of the cell, DNA fragmentation, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells (Kerr et al., 1972). Many epidemiological studies showed that protective effects of vegetables and fruits against cancer reduced risks of cardiovascular disease associated with high intakes of dietary antioxidants (Hollman and Katan, 1996).

Sennosides are the major important constituents of senna extracts from many plants, which can be transformed by bacterial enzymes to produce the sennins and anthrones before following oxidation to form anthraquinones outside the reductive atmosphere of the large intestine in humans (Kobashi et al., 1980). The sennidins can undergo further decomposition to form the aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-9,10-anthracenedione) which is a hydroxyanthraquinone in blood. From the in vitro incubation tests, it showed that aloe-emodin could induce mutagenic and genotoxic activity (Westendorf et al., 1990). But it is also reported that aloe-emodin did not induce cytotoxicity in L 1210 leukemia cells (Driscoll, 1974). Other investigators also pointed out that at high concentrations, aloe-emodin did induce cytotoxicity for erythroleukemia cell lines. The important point is that aloe-emodin had been reported to be a new type of anticancer agent with selective activity against neuroectodermal tumors (Pecere et al., 2000) and induced apoptosis in human lung squamous cell carcinoma (Lee et al., 2001) and human hepatoma cell lines (Kuo et al., 2002). So far, there is no available information which addresses the effects of aloe-emodin on apoptosis in human leukemia cells (HL-60). Thus, the present study was performed to determine whether aloe-emodin could induce apoptosis in human leukemia cells.

Section snippets

Chemicals and reagents

Aloe-emodin, trypan blue, Tris–HCl, triton X-100, propidium iodide (PI), ribonuclease A were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), potassium phosphates, and TE buffer (10 mM Tris–HCl, 1 mM EDTA, PH 7.6) were purchased from Merck Co (Darmstadt, Germany). RNase was obtained from BD Biosciences Clontech (Palo Alto, CA, USA). RPMI 1640 medium, fetal bovine serum, penicillin-streptomycin and l-glutamine were obtained from Gibco BRL (Grand Island, NY, USA).

Effects of various concentrations of aloe-emodin on cell viability of human leukemia HL-60 cells

The results from PI stain experiments indicated that <2% of HL-60 (human leukemia cells) were stained when they were incubated in RPMI 1640 medium containing 10% FBS. In contrast, in the presence of aloe-emodin (5–25 μM), the cells were increased by staining after the time and concentration increased for HL-60 cells, suggesting that aloe-emodin induced antiproliferative activity in HL-60 (Fig. 1). Further increase in the concentration of aloe-emodin resulted in greater decrease in the viable

Discussion

Leukemia is one of the most deadliest cancers in the world. Chemoprevention is a promising way to stop the development of such cancers. Large numbers of minor dietary components has been reported to inhibit various stages of carcinogenesis (Donaldson et al., 1994). Aloe-emodin has been demonstrated to be one of such chemopreventive agents but the underlying mechanisms are not well understood for HL-60 cells. Here, we have demonstrated that the possible role of the aloe-emodin was (i) to

Acknowledgments

This work was supported by grant NSC89-2745-P-039-001 from the National Science Council of Taiwan.

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Aloe-emodin induced in vitro G2/M arrest of cell cycle in human promyelocytic leukemia HL-60 cells

Abstract

Introduction

Section snippets

Chemicals and reagents

Effects of various concentrations of aloe-emodin on cell viability of human leukemia HL-60 cells

Discussion

Acknowledgments

J. Biol. Chem.

Trends Cell Biol.

Life Sci.

Euro. J. Pharmacol.

Cancer Lett.

J. Biol. Chem.

Mutat. Res.

Biochemical pathways of caspase activation during apoptosis

Ann. Rev. Cell Dev. Biol.

Activation of p34cdc2 coincident with taxol-induced apoptosis

Cell Growth Diff.

Structure-antitumor activity relationships among quinone derivatives

Cancer Chemother. Rep.

Bcl-2 family members and the mitochondria in apoptosis

Genes Dev.

Absorption, metabolism and health effects of dietary flavonoids in man

Biomed. Pharmacother.

Flow Cytometry Protocols